Apparatus and method for separating viral particles from cells

ABSTRACT

A method for analyzing a sample to detect cells infected by Human Papilloma Virus (HPV) is provided. The method includes passing a medium containing the sample across a filter. The filter has a pore size that is greater than a dimension of a HPV particle, but smaller than a dimension of a HPV infected cell, such that most of the HPV particles pass through the filter, while leaving the cells on the filter. The material collected on the filter is then examined to determine if HPV infected cells are present in the material.

FIELD OF THE INVENTION

The invention pertains to devices and methods for collecting anddetecting a biological material, and more particularly, to devices andmethods for separating biological material from viral particles.

BACKGROUND

Human Papilloma Virus (HPV) belongs to the Papovaviridae family, whichincludes double-stranded members of Papilloma viruses and polyomaviruses. HPV infects the epithelial surfaces of skin or mucosa, causinga warty growth known as condyloma. More than 100 types of HPV have beenidentified. In addition to benign warty growths, HPV may also beassociated with several types of neoplasms.

Cells infected with HPV may undergo changes in cellular morphology,typified by an apparent clearing of the cytoplasm surrounding thenucleus of the HPV infected cell. Such cellular morphology associatedwith the HPV infected cells may be observed microscopically and used fordiagnosis of HPV. Koilocytosis is the term used to describe thecytopathic effect induced by HPV infection. The observed presence ofkoilocytes in a cytological preparation of exfoliated epithelial cellsfrom the cervix provides one criterion for categorizing a cytologicdiagnosis of Low Grade Squamous Intraepithelial Lesion (LGSIL) accordingto the Bethesda Classification System. Patients classified with adiagnosis of LGSIL, a pre-neoplastic condition, are generally referredto a gynecologic oncologist for colposcopy.

As with many pathologic viruses, it is believed that HPV inside cells(intracellular HPV) is associated with the development of pre-neoplasticdisease, while HPV outside the cells (extracellular HPV) is more closelyassociated with the normal condition of the cells. Therefore, in adiagnostic procedure, it would be desirable to prepare a sample suchthat it contains no or a fewer number of extracellular HPV, and to basea diagnostic result on such prepared sample. Thus, the determination ofHPV cells will correlate more closely with a diagnosis of LGSIL thanwith a diagnosis of “Within Normal Limits” (WNL).

Various methods have been employed for detection of HPV. For example,liquid-based assay methods typically involve collection of exfoliatedepithelial cells and the surrounding extra cellular milieu, and placingthe sample into a medium, such as a detergent. The medium dissolves thesample, which is then analyzed to detect HPV. Because the mediumemployed in such method typically destroys the morphological integrityof the cells, the dissolved solution is homogeneous in that it does notallow intracellular HPV to be distinguished from extracellular HPV.

A pap smear test, which has application in the detection of early cancerof the uterine cervix, may be used to detect HPV. To perform a pap smeartest, a physician collects cells by brushing and/or scraping a skin ormucous membrane in a target area with an instrument. The cells are thensmeared onto a glass slide, and are fixed and transported to alaboratory where the slide is stained. The glass slide is then examinedunder a microscope by a cytotechnologist and/or a pathologist toidentify cellular abnormalities. During evaluation, a pathologist mayemploy a polychrome technique, characterized by staining the nuclearpart of the cells, to determine the presence of dysplasia or neoplasia.The pathologist may also apply a counter-stain for viewing the cytoplasmof the cells. Because the sample of the pap smear test may containdebris, blood, mucus, and other obscuring artifacts, the pap smear testmay be difficult to evaluate, and may not provide an accurate diagnosticassessment of the collected sample.

Cytology based on the collection of the exfoliated cervical cells into aliquid preservative offers many advantages over the traditional methodof smearing the cells directly onto the slide. A slide can be preparedfrom the cell suspension using a filter transfer technique, as disclosedin U.S. Pat. Nos. 6,572,824, 6,318,190, and 5,772,818, which areexpressly incorporated herein by reference. Debris, blood and mucus isgreatly reduced by the combined method of liquid collection andfiltering to transfer the cells onto a glass slide.

Intracellular HPV can be detected by a slide-based assay known asin-situ hybridization. In this method, a sample is first collected. Thesample is then denatured, hybridized, washed, and stained according toappropriate probe specifications. After the sample is appropriatelyprepared, it is then examined on a slide to detect HPV. Although thein-situ hybridization can be used to detect and verify intracellularHPV, it is often difficult and laborious to perform, and the result maybe open to subjective interpretation.

An alternative method for detecting HPV involves the use of the HybridCapture® System, marketed by Digene Corporation, located inGaithersburg, Md. The Hybrid Capture® System is a signal amplificationassay utilizing antibody capture and chemiluminescent signal detection.Such an assay requires clinical specimens to be combined with analkaline solution, which disrupts the virus and releases the target DNA.The target DNA then combines with specific RNA probes to create RNA:DNAhybrids, which are then captured onto a solid phase coated with captureantibodies specific for the RNA:DNA hybrids. The captured RNA:DNAhybrids are then detected with antibodies conjugated to alkalinephosphatase. Because the Hybrid Capture® method does not distinguishreleased DNA originated in extracellular HPV from released DNAoriginated in intracellular HPV, such method also does not allowintracellular HPV to be distinguished from extracellular HPV.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, a method for analyzing asample to detect cells infected by HPV is provided. The method includespassing a medium containing the sample across a filter. By way ofnon-limiting examples, the medium may be poured onto the filter, oralternatively, a pneumatic force or pressure may be used to draw themedium across the filter. In another method, the filter may be movedthrough the medium. The method may also include preparing the materialcollected on the filter for examination. The filter has a pore sizegreater than a dimension of a HPV particle but smaller than a dimensionof a HPV infected cell. For example, the pore size can range from 0.2microns to 10 microns.

Next, the material collected on the filter is examined to determine ifHPV infected cells are present in the material. Various methods may beused to prepare the collected material for examination. By way ofnon-limiting examples, the materials collected on the filter may beprepared and examined by placing the collected material onto a slide forviewing under a microscope, or by using a Hybrid Capture® method asdescribed herein. In one embodiment, the material may be placed on theslide by applying a pneumatic force across the filter. Otherconventional methods known in the art may also be used to examine thecollected materials.

According to another aspect of the invention, a method for separatingcells from extracellular HPV is provided. The method comprises passing amedium containing the cells and extracellular HPV particles across afilter to collect a majority of the cells on the filter, while passing amajority of the extracellular HPV particles through the filter. Thefilter may have the same pore characteristics described above, and themedium can be filtered in the same manner as that described above.

According to still another aspect of the invention, a method forseparating cells from extracellular HPV is provided. The methodcomprises passing a medium across the filter to collect a substance,which has an extracellular HPV to cell ratio that is substantially lessthan the extracellular HPV to cell ratio of the medium. The filter mayhave the same pore characteristics described above, and the medium canbe filtered in the same manner as that described above.

In its broadest aspects, the invention is not limited to separatingcells from extracellular HPV particles. The previously described methodscan be used to separate biological material from viral particles.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings illustrate the design and utility of preferredembodiment(s) of the invention, in which similar elements are referredto by common reference numerals. In order to better appreciate theadvantages and objects of the invention, reference should be made to theaccompanying drawings that illustrate the preferred embodiment(s). Thedrawings, however, depict the embodiment(s) of the invention, and shouldnot be taken as limiting its scope. With this caveat, the embodiment(s)of the invention will be described and explained with additionalspecificity and detail through the use of the accompanying drawings inwhich:

FIG. 1 is a flow diagram illustrating a method for analyzing a sample inaccordance with the invention;

FIG. 2A is a side view of a preferred embodiment of a system that can beused in the method of FIG. 1 to separate cells from extracellular HPVparticles;

FIG. 2B is a side view of the system of FIG. 2A, particularly shown inoperation for separating the cells from the extracellular HPV particles;

FIG. 3 is a perspective view of another preferred embodiment of a systemthat can be used in the method of FIG. 1 to separate cells fromextracellular HPV particles; and

FIG. 4 is a perspective view of yet another preferred embodiment of asystem that can be used in the method of FIG. 1 to separate cells fromextracellular HPV particles.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Referring to FIG. 1, a method 10 for determining whether a patient isinfected with intracellular HPV will now be described. Initially, asample is collected from the patient for diagnosis of a HPV condition(Step 12). The sample may be collected from any area of the patientwhere HPV can be found, such as the cervix, vulva, vagina, urethra,bladder, perianal skin, rectum, and penis. Various devices may be usedto collect the sample. In one sample collection process, a cervicalsample may be harvested by a two-stage technique, which includessampling of the endocervical canal with a cytobrush, as well asobtaining a sample from the transformation zone with a medical spatula.In another cervical sample collection process, either the cytobrush orthe spatula alone may be used. In yet another cervical sample collectionprocess, Cervex Brush&REG, available at Unimar, Inc., or AccellonCombi&REG, available at Medscand, both of which combine the action ofthe cytobrush and spatula, may be used to collect the sample. Othermethods known in the art for collecting a sample may also be used, andthe scope of the invention is not limited to a particular collectionapparatus or method.

Once a sample is collected, it is placed in a container containing aselected medium (Step 14). In one sample preservation process, themedium includes a digestive enzyme, such as, e.g., collagenase andelastase. The digestive enzyme acts as a dissolving fluid thatdisintegrates the structure of the sample, but does not affect thestructural integrity of the individual cells within the sample. Otherknown chemicals capable of performing such functions may also be used.Alternatively, the sample may be disintegrated using a tool designed oradapted for such purpose, and then placed in a selected medium. In thiscase, the medium may or may not comprise a digestive enzyme. In anothersample preservation process, the collected sample may be placed in apreservative fluid for subsequent use.

Next, the medium, which contains the disintegrated sample, is passedthrough a membrane or filter having a plurality of pores (Step 16). Thepores of the filter are sized such that they prevent cells, such ascells infected with HPV, from passing through, while allowing most orall of the extracellular HPV particles to pass through. For example, afilter having an 8 micron nominal pore size is employed in oneembodiment. Since HPV particles have a dimension of about 50 nanometers,extracellular HPV particles will pass through the filter if they arepresent in the sample. In contrast, epithelial cells have a crosssectional dimension that are generally greater than 8 microns.Therefore, if HPV infected cells are present in the sample, they will beretained on the filter without passing through it. In alternativeembodiments, a filter having a pore size that is within the range of 0.2micron to 12 microns may also be used.

The minimum number of HPV genomes, each viral particle containing onegenome, that is required to provide a positive result for the HybridCapture 2 assay is about 11,400. This number can be calculated from 0.05pg of HPV DNA that is used in each Hybrid Capture assay to determinepositive or negative specimen result in the assay and the fact that theHPV genome has 8000 base pairs. Therefore, if fewer than 11,400 HPVparticles remain with the cells after filtration, the specimen isconsidered negative for HPV by the Hybrid Capture 2 method. Cells thathave been cultured from cervical cancer specimens range from 2 HPVgenomes per cell (the SiHa cell line) to 600 genomes per cell (CaSkicell line). A positive specimen by Hybrid Capture 2 would require 600SiHa cells but only 2 CaSki cells.

As a result, material collected on the filter will have an extracellularHPV to cell ratio (i.e., the number of extracellular HPV particles overthe number of cells) that is substantially reduced from theextracellular HPV to cell ratio of the unfiltered sample. Thecorrelation between a diagnosis of intracellular HPV and an actualoccurrence of intracellular HPV will be increased by examining thecollected material rather than the unfiltered sample.

The filter may be made from a variety of materials such aspolycarbonate. For example, polycarbonate membrane marketed by theNuclepore Corporation in Pleasanton, Calif., may be used. Other filterscan be formed from materials including cellulose, nylon, polyester,Teflon®, or any other suitable material. Further details regarding theconstruction and use of such filters are disclosed in U.S. Pat. Nos.5,364,597 and 5,942,700, the entire disclosures of which are expresslyincorporated by reference herein.

Various methods may be used to pass the medium through the filter. Inone method, filtering may be accomplished by a filter transfer method.FIG. 2A illustrates a system 50 that may be used to perform a filtertransfer method. The system 50 includes a container 52, a fluidsuspension of dispersed particles 54, a particle collection device 56, afilter 58, a cap 60, a conduit 62, a pneumatic source 64, and aprocessor 68. The container 52 holds the fluid sample 54 and makes itavailable for sampling by the collection device 56. The collectiondevice 56 may be a filter cylinder device manufactured and marketed bythe Cytyc Corporation of Boxboro, Mass. In the illustrated embodiment,the fluid sample 54 is the medium that contains the collected sample,and the filter 58 is the filter, as described previously with referenceto step 16 of the method 10.

In one filtering process, the system 50 rapidly rotates the particlecollection device 56 to actuate the fluid sample 54 and disintegrateclumps of particles that may exist within the fluid sample 54. Forexample, with a fluid sample 54 having contained therein a population ofepithelial cells, the actuation of the collection device 56 candisintegrate clumped cells, such that there exists within the fluidsample 54 a population of individual cells, and a reduced population ofclumped cells. Alternatively, if the clumped cells are alreadydisintegrated, i.e., by a dissolving medium, as discussed previously,then this step may not be necessary.

The system 50 uses a pneumatic particle collection technique, whereinpneumatic action is provided by the pneumatic source 64 to draw aportion of the fluid sample 54 past the filter 58 and into thecollection device 56. FIG. 2B depicts the system 50 having drawn aportion of the sample fluid 54 across the filter 58 and into theinterior of the collection device 56. The pneumatic source 64 creates anegative pressure within the interior of the collection device 56, whichgenerates a flow of the fluid sample 54 across the filter 58. Drawing afluid sample 54 across the filter 58 causes cells dispersed within thefluid sample 54 to collect against the filter 58 and, in particular, toblock the pores of the filter membrane. Viral particles, such asextracellular HPV particles, if they are present in the fluid 54, willeither pass through the pores of the filter 58 as the fluid 54 is drawnacross the filter 58, or remain in the fluid 54.

During filtering of the sample, the processor 68 determines a measurerepresentative of the quantity of cells that have collected against thefilter 58. The action of blocking the pores of the filter membrane 58 isunderstood to effectively decrease the porosity of the filter membrane58. That is, as cells collect against the filter surface 58, the poresof the filter 58 are sealed, thereby reducing the number of poresavailable for passing fluid to the interior of the collection device 56.The amount of time it takes for the negative pressure to return toequilibrium after the pneumatic source has changed the interior pressurewithin the collection device 56 is dependent, in part, on the number ofpores of filter 58 available for passing fluid into the interior of thecollection device 56. The reduction of available pores increases theamount of time it takes for the vacuum inside the collection device 56to return to equilibrium. Further, the rate of pressure change withinthe collection device 56 changes as pores are blocked. Accordingly, thepressure change and rate of pressure change within the collection device56 is representative of the number of cells that have collected againstthe surface of the filter 58. The processor 68 can track the pressurewithin the collection device 56 and determine, responsive thereto, anumber representative of a quantity of cells collected against thefilter 58. Therefore, upon extraction of the collection device 56 fromthe sample 54, the system 50 has collected a substantially knownquantity of cells from the fluid sample 54. Such filter transfer methodsare described in U.S. Pat. No. 5,942,700, which was previously mentionedherein.

In another filtering process, as shown in FIG. 3, the medium containingthe sample (i.e., medium 100) is simply poured over a filter 102. Thefilter 102 is described previously with reference to step 16. Most orall of the extracellular HPV, if present, will pass through the pores ofthe filter 102 together with the medium 100, while cells are retained bythe filter 102, as discussed previously. A separate container 104 may beused to contain the medium 100 that has passed through the filter 102.

In yet another filtering process, the filter 102 is moved over themedium 100. For example, the filter 102 may be placed at a bottom of acontainer 110, and the medium 100 containing the dissolved ordisintegrated sample is then poured into the container 110 (FIG. 4).Alternatively, instead of disintegrating the sample first, the samplemay be placed over the filter 102 in the container 110, and the medium100, which contains a digestive enzyme, is then poured into thecontainer 110 to dissolve the sample. Next, the filter 102 is movedthrough the medium 100, collecting HPV infected cells that may bepresent in the medium 100, while leaving most or all of theextracellular HPV in the medium 100.

Optionally, the sample may be further filtered by one or more additionalfilters, which may have a larger or smaller pore size than the firstfilter 102. For example, further filtering using a filter that haslarger pore size may be beneficial in that it allows any artifacts thatare larger than HPV infected cells to be separated from the HPV infectedcells, which may or may not be present in the collected sample. In thiscase, materials collected on the first filter may be placed in anothermedium, which is then caused to pass through the additional filter. Anyof the above described filtering techniques may be used to perform suchfurther filtering.

It should be noted that a prerequisite of viral internalization into thecell is binding of the viral particle to the cell surface. Cell surfacetargets for a variety of pathogenic organisms have been identified. Anexample is cell-surface hyaluronic acid as a receptor target forChlamydia trachomatis. The specific cell-surface target for HPV has notbeen identified specifically, however it may be an epithelial cellspecific target since other cells types (i.e. neural, endothelial,stromal) have not been characterized to contain HPV. HPV bound to theepithelial cell surface, but not yet internalized by the cell, mayaffect the results of the filtration method described herein as long asmore than about 11,400 HPV particles remain adhered to the cells. InSitu hybridization studies have not revealed HPV particles bound to theexterior of the cell, however the process of In Situ hybridization isrigorous and involves treating the cell specimen with enzyme, manyreagents, temperature changes and washing steps that may scour thesurface of the cells of any bound virus particles.

Returning to FIG. 1, after the material is collected on the filter, thematerial is prepared for subsequent examination (Step 18). If theoriginally collected sample contains intracellular HPV and extracellularHPV, the HPV infected cells will have been collected and separated fromthe extracellular HPV. Various techniques known in the art may be usedto prepare the materials collected on the filter for examination. In onesample preparation process, a slide is prepared from the materialscollected on the filter for subsequent examination using a conventionaltechnique.

Alternatively, a ThinPrep® slide may be prepared by placing a uniformsingle layer of the collected material onto a glass slide. This can beaccomplished by placing the outer surface of the filter on which thematerials are collected onto a slide. Then a pneumatic pressure isapplied across the filter to dislodge the collected materials from theouter surface of the filter and urge the dislodged material against theslide. The final product will essentially contain a monolayer of cellsfree of debris, blood, mucus, and other obscuring artifacts.Alternatively, the materials collected on the filter may be transferredto another container, which can then be used to prepare a ThinPrep®slide, as similarly discussed previously. Systems and methods forpreparing ThinPrep® slides are described in U.S. Pat. Nos. 5,143,627 and5,364,597.

After the sample preparation process has been completed, the preparedsample is then examined to determine if intracellular HPV is present(step 20). For example, the collected materials may be examined under amicroscope, which may be automated such as that embodied in the ThinPrepImaging System, marketed by Cytyc Corporation.

Other methods known in the art for examining cells may also be used. Forexample, the collected material may be examined to detect intracellularHPV using the Hybrid Capture® method described previously. In this case,instead of making a slide, the collected material is deposited into avial containing a volume of cell lysing solution, such as an alkalinesolution, which disrupts the virus and releases the target DNA. Thetarget DNA then combines with specific RNA probes to create RNA:DNAhybrids, which are then captured onto a solid phase coated with captureantibodies specific for the RNA:DNA hybrids. The captured RNA:DNAhybrids are then detected with antibodies conjugated to alkalinephosphatase. The bound alkaline phosphatase is detected with achemiluminescent dioxetane substrate. Upon cleavage by the alkalinephosphatase, the substrate produces light that is measured on aluminometer in Relative Light Units (RLUs). If the originally collectedsample contains intracellular HPV, the output of the luminometer willindicate so. An example of an assay that utilizes antibody capture andchemiluminescent signal detection is the HC2 HPV Assay, available atDigene Corporation, located in Gaithersburg, Md.

Although several embodiments of the method have been described withreference to separating HPV infected cells from extracellular HPV, themethod may also be used to separate different materials in a sample indifferent applications. For example, the method may be used in otherclinical or laboratory applications, where it may desirable to collector detect a biological material from a sample. In one method, thefiltering process described in step 16 may be used to separate cellsfrom extracellular viral particles other than HPV, such as Adenovirus orEpstein Barr Virus. Furthermore, the method may also be used to separatea biological material, such as a component of a cell, from a viralparticle. The method may also be used to separate cell types by theirsize.

Thus, although different embodiments have been shown and described, itwould be apparent to those skilled in the art that many changes andmodifications may be made thereunto without the departing from the scopeof the invention, which is defined by the following claims and theirequivalents.

1. A method for analyzing a biological sample to detect the presence orabsence of cells infected by human papilloma virus (HPV), comprising thesteps of: obtaining a sample from a patient that may comprise cellsinfected by HPV; placing said sample in a liquid medium; passing saidmedium containing said sample across a filter to collect cells from saidmedium on said filter, said filter having a pore size that is greaterthan a dimension of a HPV particle but smaller than a dimension of a HPVinfected cell; and examining said collected cells to determine if HPVinfected cells are present.
 2. The method of claim 1, wherein said poresize is within a range of 0.2 micron to 10 microns.
 3. The method ofclaim 1, wherein said pore size is approximately 8 microns.
 4. Themethod of claim 1, wherein said cells are epithelial cells.
 5. Themethod of claim 1, wherein said cells are cervical cells.